Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53

ABSTRACT

The invention relates a method wherein a molecule is used for inducing and/or promoting skipping of at least one of exon 43, exon 46, exons 50-53 of the DMD pre-mRNA in a patient, preferably in an isolated cell of a patient, the method comprising providing the cell and/or the patient with a molecule. The invention also relates to the molecule as such.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/289,053 filed on Oct. 7, 2016 which is a continuation of U.S.application Ser. No. 14/631,686 filed on Feb. 25, 2015, now U.S. Pat.No. 9,499,818, issued Nov. 22, 2016, which is a continuation of U.S.application Ser. No. 13/094,571 filed Apr. 26, 2011, which is acontinuation of International Application No. PCT/NL2009/050113, filedon Mar. 11, 2009, which is a continuation of PCT/NL2008/050673, filed onOct. 27, 2008, the contents of each of which are herein incorporated byreference in their entirety.

REFERENCE TO A SEQUENCE LISTING

The present specification is being filed with a Sequence Listing inComputer Readable Form (CFR), which is entitled11808-364-999_SEQLIST.txt of 128,829 bytes in size and was created Nov.15, 2018; the content of which is incorporated herein by reference inits entirety.

FIELD

The invention relates to the field of genetics, more specifically humangenetics. The invention in particular relates to modulation of splicingof the human Duchenne Muscular Dystrophy pre-mRNA.

BACKGROUND

Myopathies are disorders that result in functional impairment ofmuscles. Muscular dystrophy (MD) refers to genetic diseases that arecharacterized by progressive weakness and degeneration of skeletalmuscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy(BMD) are the most common childhood forms of muscular dystrophy. Theyare recessive disorders and because the gene responsible for DMD and BMDresides on the X-chromosome, mutations mainly affect males with anincidence of about 1 in 3500 boys.

DMD and BMD are caused by genetic defects in the DMD gene encodingdystrophin, a muscle protein that is required for interactions betweenthe cytoskeleton and the extracellular matrix to maintain muscle fiberstability during contraction. DMD is a severe, lethal neuromusculardisorder resulting in a dependency on wheelchair support before the ageof 12 and DMD patients often die before the age of thirty due torespiratory- or heart failure. In contrast, BMD patients often remainambulatory until later in life, and have near normal life expectancies.DMD mutations in the DMD gene are characterized by frame shiftinginsertions or deletions or nonsense point mutations, resulting in theabsence of functional dystrophin. BMD mutations in general keep thereading frame intact, allowing synthesis of a partly functionaldystrophin.

During the last decade, specific modification of splicing in order torestore the disrupted reading frame of the dystrophin transcript hasemerged as a promising therapy for Duchenne muscular dystrophy (DMD)(van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008;10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, vanDeutekom et al., N Engl J Med. 2007; 357(26):2677-86).

Using antisense oligonucleotides (AONs) interfering with splicingsignals the skipping of specific exons can be induced in the DMDpre-mRNA, thus restoring the open reading frame and converting thesevere DMD into a milder BMD phenotype (van Deutekom et al. Hum MolGenet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003;12(8):907-14.). In vivo proof-of-concept was first obtained in the mdxmouse model, which is dystrophin-deficient due to a nonsense mutation inexon 23. Intramuscular and intravenous injections of AONs targeting themutated exon 23 restored dystrophin expression for at least three months(Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA.2005; 102(1):198-203). This was accompanied by restoration ofdystrophin-associated proteins at the fiber membrane as well asfunctional improvement of the treated muscle. In vivo skipping of humanexons has also been achieved in the hDMD mouse model, which contains acomplete copy of the human DMD gene integrated in chromosome 5 of themouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; ′t Hoenet al. J Biol Chem. 2008; 283: 5899-907).

Recently, a first-in-man study was successfully completed where an AONinducing the skipping of exon 51 was injected into a small area of thetibialis anterior muscle of four DMD patients. Novel dystrophinexpression was observed in the majority of muscle fibers in all fourpatients treated, and the AON was safe and well tolerated (van Deutekomet al. N Engl J Med. 2007; 357: 2677-86).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In human control myotubes, a series of AONs (PS237, PS238, andPS240; SEQ ID NO 65, 66, 16 respectively) targeting exon 43 was testedat 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels ofexon 43 skipping. (M: DNA size marker; NT: non-treated cells)

FIG. 2. In myotubes from a DMD patient with an exon 45 deletion, aseries of AONs (PS177, PS179, PS181, and PS182; SEQ ID NO 91, 70, 110,and 117 respectively) targeting exon 46 was tested at two differentconcentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproduciblyinduced highest levels of exon 46 skipping. (M: DNA size marker)

FIG. 3. In human control myotubes, a series of AONs (PS245, PS246,PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively)targeting exon 50 was tested at 500 nM. PS248 (SEQ ID NO 127)reproducibly induced highest levels of exon 50 skipping. (M: DNA sizemarker; NT: non-treated cells).

FIG. 4. In human control myotubes, two novel AONs (PS232 and PS236; SEQID NO 246 and 299 respectively) targeting exon 52 were tested at twodifferent concentrations (200 and 500 nM) and directly compared to apreviously described AON (52-1). PS236 (SEQ ID NO 299) reproduciblyinduced highest levels of exon 52 skipping. (M: DNA size marker; NT:non-treated cells).

DETAILED DESCRIPTION

Method

In a first aspect, the present invention provides a method for inducing,and/or promoting skipping of at least one of exons 43, 46, 50-53 of theDMD pre-mRNA in a patient, preferably in an isolated cell of a patient,the method comprising providing said cell and/or said patient with amolecule that binds to a continuous stretch of at least 8 nucleotideswithin said exon. It is to be understood that said method encompasses anin vitro, in vivo or ex vivo method.

Accordingly, a method is provided for inducing and/or promoting skippingof at least one of exons 43, 46, 50-53 of DMD pre-mRNA in a patient,preferably in an isolated cell of said patient, the method comprisingproviding said cell and/or said patient with a molecule that binds to acontinuous stretch of at least 8 nucleotides within said exon.

As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMDgene of a DMD or BMD patient.

A patient is preferably intended to mean a patient having DMD or BMD aslater defined herein or a patient susceptible to develop DMD or BMD dueto his or her genetic background. In the case of a DMD patient, anoligonucleotide used will preferably correct one mutation as present inthe DMD gene of said patient and therefore will preferably create a DMDprotein that will look like a BMD protein: said protein will preferablybe a functional dystrophin as later defined herein. In the case of a BMDpatient, an oligonucleotide as used will preferably correct one mutationas present in the BMD gene of said patient and therefore will preferablycreate a dystrophin which will be more functional than the dystrophinwhich was originally present in said BMD patient.

Exon skipping refers to the induction in a cell of a mature mRNA thatdoes not contain a particular exon that is normally present therein.Exon skipping is performed by providing a cell expressing the pre-mRNAof said mRNA with a molecule capable of interfering with essentialsequences such as for example the splice donor of splice acceptorsequence that required for splicing of said exon, or a molecule that iscapable of interfering with an exon inclusion signal that is requiredfor recognition of a stretch of nucleotides as an exon to be included inthe mRNA. The term pre-mRNA refers to a non-processed or partlyprocessed precursor mRNA that is synthesized from a DNA template in thecell nucleus by transcription.

Within the context of the invention, inducing and/or promoting skippingof an exon as indicated herein means that at least 1%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more(muscle) cells of a treated patient will not contain said exon. This ispreferably assessed by PCR as described in the examples.

Preferably, a method of the invention by inducing and/or promotingskipping of at least one of the following exons 43, 46, 50-53 of the DMDpre-mRNA in one or more (muscle) cells of a patient, provides saidpatient with a functional dystrophin protein and/or decreases theproduction of an aberrant dystrophin protein in said patient and/orincreases the production of a functional dystrophin is said patient.Providing a patient with a functional dystrophin protein and/ordecreasing the production of an aberrant dystrophin protein in saidpatient is typically applied in a DMD patient. Increasing the productionof a functional dystrophin is typically applied in a BMD patient.

Therefore a preferred method is a method, wherein a patient or one ormore cells of said patient is provided with a functional dystrophinprotein and/or wherein the production of an aberrant dystrophin proteinin said patient is decreased and/or wherein the production of afunctional dystrophin is increased in said patient, wherein the level ofsaid aberrant or functional dystrophin is assessed by comparison to thelevel of said dystrophin in said patient at the onset of the method.

Decreasing the production of an aberrant dystrophin may be assessed atthe mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrantdystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophinmRNA or protein is also referred to herein as a non-functionaldystrophin mRNA or protein. A non functional dystrophin protein ispreferably a dystrophin protein which is not able to bind actin and/ormembers of the DGC protein complex. A non-functional dystrophin proteinor dystrophin mRNA does typically not have, or does not encode adystrophin protein with an intact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the mRNA level (by RT-PCRanalysis) and preferably means that a detectable amount of a functionaldystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectabledystrophin mRNA is a functional dystrophin mRNA.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the protein level (byimmunofluorescence and western blot analyses) and preferably means thata detectable amount of a functional dystrophin protein is detectable byimmunofluorescence or western blot analysis. In another embodiment, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of thedetectable dystrophin protein is a functional dystrophin protein.

As defined herein, a functional dystrophin is preferably a wild typedystrophin corresponding to a protein having the amino acid sequence asidentified in SEQ ID NO: 1. A functional dystrophin is preferably adystrophin, which has an actin binding domain in its N terminal part(first 240 amino acids at the N terminus), a cystein-rich domain (aminoacid 3361 till 3685) and a C terminal domain (last 325 amino acids atthe C terminus) each of these domains being present in a wild typedystrophin as known to the skilled person. The amino acids indicatedherein correspond to amino acids of the wild type dystrophin beingrepresented by SEQ ID NO:1. In other words, a functional dystrophin is adystrophin which exhibits at least to some extent an activity of a wildtype dystrophin. “At least to some extent” preferably means at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of acorresponding activity of a wild type functional dystrophin. In thiscontext, an activity of a functional dystrophin is preferably binding toactin and to the dystrophin-associated glycoprotein complex (DGC)(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Binding of dystrophin to actin and to the DGC complex may bevisualized by either co-immunoprecipitation using total protein extractsor immunofluorescence analysis of cross-sections, from a muscle biopsy,as known to the skilled person.

Individuals or patients suffering from Duchenne muscular dystrophytypically have a mutation in the gene encoding dystrophin that preventsynthesis of the complete protein, i.e of a premature stop prevents thesynthesis of the C-terminus. In Becker muscular dystrophy the DMD genealso comprises a mutation compared tot the wild type gene but themutation does typically not induce a premature stop and the C-terminusis typically synthesized. As a result a functional dystrophin protein issynthesized that has at least the same activity in kind as the wild typeprotein, not although not necessarily the same amount of activity. Thegenome of a BMD individual typically encodes a dystrophin proteincomprising the N terminal part (first 240 amino acids at the Nterminus), a cystein-rich domain (amino acid 3361 till 3685) and a Cterminal domain (last 325 amino acids at the C terminus) but its centralrod shaped domain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Exon skipping for the treatment of DMD is typically directedto overcome a premature stop in the pre-mRNA by skipping an exon in therod-shaped domain to correct the reading frame and allow synthesis ofremainder of the dystrophin protein including the C-terminus, albeitthat the protein is somewhat smaller as a result of a smaller roddomain. In a preferred embodiment, an individual having DMD and beingtreated by a method as defined herein will be provided a dystrophinwhich exhibits at least to some extent an activity of a wild typedystrophin. More preferably, if said individual is a Duchenne patient oris suspected to be a Duchenne patient, a functional dystrophin is adystrophin of an individual having BMD: typically said dystrophin isable to interact with both actin and the DGC, but its central rod shapeddomain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). The central rod-shaped domain of wild type dystrophincomprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entriesin the leiden Duchenne Muscular Dystrophy mutation database: an overviewof mutation types and paradoxical cases that confirm the reading-framerule, Muscle Nerve, 34: 135-144). For example, a central rod-shapeddomain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22or 12 to 18 spectrin-like repeats as long as it can bind to actin and toDGC.

A method of the invention may alleviate one or more characteristics of amyogenic or muscle cell of a patient or alleviate one or more symptomsof a DMD patient having a deletion including but not limited to exons44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping),13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52(correctable by exon 51 skipping), exons 8-51, 51, 53, 53-55, 53-57,53-59, 53-60, (correctable by exon 52 skipping) and exons 10-52, 42-52,43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53skipping) in the DMD gene, occurring in a total of 68% of all DMDpatients with a deletion (Aartsma-Rus et al., Hum. Mut. 2009).

Alternatively, a method of the invention may improve one or morecharacteristics of a muscle cell of a patient or alleviate one or moresymptoms of a DMD patient having small mutations in, or single exonduplications of exon 43, 46, 50-53 in the DMD gene, occurring in a totalof 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut.2009)

Furthermore, for some patients the simultaneous skipping of one of moreexons in addition to exon 43, exon 46 and/or exon 50-53 is required torestore the open reading frame, including patients with specificdeletions, small (point) mutations, or double or multiple exonduplications, such as (but not limited to) a deletion of exons 44-50requiring the co-skipping of exons 43 and 51, with a deletion of exons46-50 requiring the co-skipping of exons 45 and 51, with a deletion ofexons 44-52 requiring the co-skipping of exons 43 and 53, with adeletion of exons 46-52 requiring the co-skipping of exons 45 and 53,with a deletion of exons 51-54 requiring the co-skipping of exons 50 and55, with a deletion of exons 53-54 requiring the co-skipping of exons 52and 55, with a deletion of exons 53-56 requiring the co-skipping ofexons 52 and 57, with a nonsense mutation in exon 43 or exon 44requiring the co-skipping of exon 43 and 44, with a nonsense mutation inexon 45 or exon 46 requiring the co-skipping of exon 45 and 46, with anonsense mutation in exon 50 or exon 51 requiring the co-skipping ofexon 50 and 51, with a nonsense mutation in exon 51 or exon 52 requiringthe co-skipping of exon 51 and 52, with a nonsense mutation in exon 52or exon 53 requiring the co-skipping of exon 52 and 53, or with a doubleor multiple exon duplication involving exons 43, 46, 50, 51, 52, and/or53.

In a preferred method, the skipping of exon 43 is induced, or theskipping of exon 46 is induced, or the skipping of exon 50 is induced orthe skipping of exon 51 is induced or the skipping of exon 52 is inducedor the skipping of exon 53 is induced. An induction of the skipping oftwo of these exons is also encompassed by a method of the invention. Forexample, preferably skipping of exons 50 and 51, or 52 and 53, or 43 and51, or 43 and 53, or 51 and 52. Depending on the type and the identity(the specific exons involved) of mutation identified in a patient, theskilled person will know which combination of exons needs to be skippedin said patient.

In a preferred method, one or more symptom(s) of a DMD or a BMD patientis/are alleviated and/or one or more characteristic(s) of one or moremuscle cells from a DMD or a BMD patient is/are improved. Such symptomsor characteristics may be assessed at the cellular, tissue level or onthe patient self.

An alleviation of one or more characteristics may be assessed by any ofthe following assays on a myogenic cell or muscle cell from a patient:reduced calcium uptake by muscle cells, decreased collagen synthesis,altered morphology, altered lipid biosynthesis, decreased oxidativestress, and/or improved muscle fiber function, integrity, and/orsurvival. These parameters are usually assessed using immunofluorescenceand/or histochemical analyses of cross sections of muscle biopsies.

The improvement of muscle fiber function, integrity and/or survival maybe assessed using at least one of the following assays: a detectabledecrease of creatine kinase in blood, a detectable decrease of necrosisof muscle fibers in a biopsy cross-section of a muscle suspected to bedystrophic, and/or a detectable increase of the homogeneity of thediameter of muscle fibers in a biopsy cross-section of a musclesuspected to be dystrophic. Each of these assays is known to the skilledperson.

Creatine kinase may be detected in blood as described in Hodgetts et al(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).A detectable decrease in creatine kinase may mean a decrease of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to theconcentration of creatine kinase in a same DMD or BMD patient beforetreatment.

A detectable decrease of necrosis of muscle fibers is preferablyassessed in a muscle biopsy, more preferably as described in Hodgetts etal (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16:591-602.2006) using biopsy cross-sections. A detectable decrease ofnecrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more of the area wherein necrosis has been identified usingbiopsy cross-sections. The decrease is measured by comparison to thenecrosis as assessed in a same DMD or BMD patient before treatment.

A detectable increase of the homogeneity of the diameter of a musclefiber is preferably assessed in a muscle biopsy cross-section, morepreferably as described in Hodgetts et al (Hodgetts S., et al, (2006),Neuromuscular Disorders, 16: 591-602.2006). The increase is measured bycomparison to the homogeneity of the diameter of a muscle fiber in asame DMD or BMD patient before treatment. An alleviation of one or moresymptoms may be assessed by any of the following assays on the patientself: prolongation of time to loss of walking, improvement of musclestrength, improvement of the ability to lift weight, improvement of thetime taken to rise from the floor, improvement in the nine-meter walkingtime, improvement in the time taken for four-stairs climbing,improvement of the leg function grade, improvement of the pulmonaryfunction, improvement of cardiac function, improvement of the quality oflife. Each of these assays is known to the skilled person. As anexample, the publication of Manzur at al (Manzur A Y et al, (2008),Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review),Wiley publishers, The Cochrane collaboration.) gives an extensiveexplanation of each of these assays. For each of these assays, as soonas a detectable improvement or prolongation of a parameter measured inan assay has been found, it will preferably mean that one or moresymptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy hasbeen alleviated in an individual using a method of the invention.Detectable improvement or prolongation is preferably a statisticallysignificant improvement or prolongation as described in Hodgetts et al(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).Alternatively, the alleviation of one or more symptom(s) of DuchenneMuscular Dystrophy or Becker Muscular Dystrophy may be assessed bymeasuring an improvement of a muscle fiber function, integrity and/orsurvival as later defined herein.

A treatment in a method according to the invention may have a durationof at least one week, at least one month, at least several months, atleast one year, at least 2, 3, 4, 5, 6 years or more.

Each molecule or oligonucleotide or equivalent thereof as defined hereinfor use according to the invention may be suitable for directadministration to a cell, tissue and/or an organ in vivo of individualsaffected by or at risk of developing DMD or BMD, and may be administereddirectly in vivo, ex vivo or in vitro. The frequency of administrationof a molecule or an oligonucleotide or a composition of the inventionmay depend on several parameters such as the age of the patient, themutation of the patient, the number of molecules (dose), the formulationof said molecule. The frequency may be ranged between at least once in atwo weeks, or three weeks or four weeks or five weeks or a longer timeperiod.

A molecule or oligonucleotide or equivalent thereof can be delivered asis to a cell. When administering said molecule, oligonucleotide orequivalent thereof to an individual, it is preferred that it isdissolved in a solution that is compatible with the delivery method. Forintravenous, subcutaneous, intramuscular, intrathecal and/orintraventricular administration it is preferred that the solution is aphysiological salt solution. Particularly preferred for a method of theinvention is the use of an excipient that will further enhance deliveryof said molecule, oligonucleotide or functional equivalent thereof asdefined herein, to a cell and into a cell, preferably a muscle cell.Preferred excipient are defined in the section entitled “pharmaceuticalcomposition”.

In a preferred method of the invention, an additional molecule is usedwhich is able to induce and/or promote skipping of another exon of theDMD pre-mRNA of a patient. Preferably, the second exon is selected from:exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62,63, 65, 66, 69, or 75 of the DMD pre-mRNA of a patient. Molecules whichcan be used are depicted in any one of Table 1 to 7. This way, inclusionof two or more exons of a DMD pre-mRNA in mRNA produced from thispre-mRNA is prevented. This embodiment is further referred to as double-or multiexon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al.Antisense-induced multiexon skipping for Duchenne muscular dystrophymakes more sense. Am J Hum Genet 2004; 74(1):83-92, Aartsma-Rus A, KamanW E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploringthe frontiers of therapeutic exon skipping for Duchenne musculardystrophy by double targeting within one or multiple exons. Mol Ther2006; 14(3):401-7). In most cases double-exon skipping results in theexclusion of only the two targeted exons from the DMD pre-mRNA. However,in other cases it was found that the targeted exons and the entireregion in between said exons in said pre-mRNA were not present in theproduced mRNA even when other exons (intervening exons) were present insuch region. This multi-skipping was notably so for the combination ofoligonucleotides derived from the DMD gene, wherein one oligonucleotidefor exon 45 and one oligonucleotide for exon 51 was added to a celltranscribing the DMD gene. Such a set-up resulted in mRNA being producedthat did not contain exons 45 to 51. Apparently, the structure of thepre-mRNA in the presence of the mentioned oligonucleotides was such thatthe splicing machinery was stimulated to connect exons 44 and 52 to eachother.

It is possible to specifically promote the skipping of also theintervening exons by providing a linkage between the two complementaryoligonucleotides. Hence, in one embodiment stretches of nucleotidescomplementary to at least two dystrophin exons are separated by alinking moiety. The at least two stretches of nucleotides are thuslinked in this embodiment so as to form a single molecule.

In case, more than one compounds or molecules are used in a method ofthe invention, said compounds can be administered to an individual inany order. In one embodiment, said compounds are administeredsimultaneously (meaning that said compounds are administered within 10hours, preferably within one hour). This is however not necessary. Inanother embodiment, said compounds are administered sequentially.

Molecule

In a second aspect, there is provided a molecule for use in a method asdescribed in the previous section entitled “Method”. A molecule asdefined herein is preferably an oligonucleotide or antisenseoligonucleotide (AON).

It was found by the present investigators that any of exon 43, 46, 50-53is specifically skipped at a high frequency using a molecule thatpreferably binds to a continuous stretch of at least 8 nucleotideswithin said exon. Although this effect can be associated with a higherbinding affinity of said molecule, compared to a molecule that binds toa continuous stretch of less than 8 nucleotides, there could be otherintracellular parameters involved that favor thermodynamic, kinetic, orstructural characteristics of the hybrid duplex. In a preferredembodiment, a molecule that binds to a continuous stretch of at least 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50 nucleotides within said exon is used.

In a preferred embodiment, a molecule or an oligonucleotide of theinvention which comprises a sequence that is complementary to a part ofany of exon 43, 46, 50-53 of DMD pre-mRNA is such that the complementarypart is at least 50% of the length of the oligonucleotide of theinvention, more preferably at least 60%, even more preferably at least70%, even more preferably at least 80%, even more preferably at least90% or even more preferably at least 95%, or even more preferably 98%and most preferably up to 100%. “A part of said exon” preferably means astretch of at least 8 nucleotides. In a most preferred embodiment, anoligonucleotide of the invention consists of a sequence that iscomplementary to part of said exon DMD pre-mRNA as defined herein. Forexample, an oligonucleotide may comprise a sequence that iscomplementary to part of said exon DMD pre-mRNA as defined herein andadditional flanking sequences. In a more preferred embodiment, thelength of said complementary part of said oligonucleotide is of at least8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flankingsequences are used to modify the binding of a protein to said moleculeor oligonucleotide, or to modify a thermodynamic property of theoligonucleotide, more preferably to modify target RNA binding affinity.

A preferred molecule to be used in a method of the invention binds or iscomplementary to a continuous stretch of at least 8 nucleotides withinone of the following nucleotide sequences selected from:

(SEQ ID NO: 2) 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ for skipping of exon 43;(SEQ ID NO: 3) 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ for skipping of exon 46;(SEQ ID NO: 4) 5′-GGCGGTAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACCUAGC UCCUGGACUGACCACUAUUGG-3′ for skipping of exon 50; (SEQ ID NO: 5)5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′for skipping of exon 51; (SEQ ID NO: 6)5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′for skipping of exon 52, and (SEQ ID NO: 7)5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ for skipping of exon 53.

Of the numerous molecules that theoretically can be prepared to bind tothe continuous nucleotide stretches as defined by SEQ ID NO 2-7 withinone of said exons, the invention provides distinct molecules that can beused in a method for efficiently skipping of at least one of exon 43,exon 46 and/or exon 50-53. Although the skipping effect can be addressedto the relatively high density of putative SR protein binding siteswithin said stretches, there could be other parameters involved thatfavor uptake of the molecule or other, intracellular parameters such asthermodynamic, kinetic, or structural characteristics of the hybridduplex.

It was found that a molecule that binds to a continuous stretchcomprised within or consisting of any of SEQ ID NO 2-7 results in highlyefficient skipping of exon 43, exon 46 and/or exon 50-53 respectively ina cell and/or in a patient provided with this molecule. Therefore, in apreferred embodiment, a method is provided wherein a molecule binds to acontinuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40,45, 50 nucleotides within SEQ ID NO 2-7.

In a preferred embodiment for inducing and/or promoting the skipping ofany of exon 43, exon 46 and/or exon 50-53, the invention provides amolecule comprising or consisting of an antisense nucleotide sequenceselected from the antisense nucleotide sequences depicted in any ofTables 1 to 6. A molecule of the invention preferably comprises orconsist of the antisense nucleotide sequence of SEQ ID NO 16, SEQ ID NO65, SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ IDNO 299, SEQ ID NO:357.

A preferred molecule of the invention comprises a nucleotide-based ornucleotide or an antisense oligonucleotide sequence of between 8 and 50nucleotides or bases, more preferred between 10 and 50 nucleotides, morepreferred between 20 and 40 nucleotides, more preferred between 20 and30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides,23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides. A mostpreferred molecule of the invention comprises a nucleotide-basedsequence of 25 nucleotides.

Furthermore, none of the indicated sequences is derived from conservedparts of splice junction sites. Therefore, said molecule is not likelyto mediate differential splicing of other exons from the DMD pre-mRNA orexons from other genes.

In one embodiment, a molecule of the invention is a compound moleculethat binds to the specified sequence, or a protein such as anRNA-binding protein or a non-natural zinc-finger protein that has beenmodified to be able to bind to the corresponding nucleotide sequence ona DMD pre-RNA molecule. Methods for screening compound molecules thatbind specific nucleotide sequences are, for example, disclosed inPCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are hereinincorporated by reference. Methods for designing RNA-binding Zinc-fingerproteins that bind specific nucleotide sequences are disclosed byFriesen and Darby, Nature Structural Biology 5: 543-546 (1998) which isherein incorporated by reference.

A preferred molecule of the invention binds to at least part of thesequence of SEQ ID NO 2:5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ which is present in exon 43 of theDMD gene. More preferably, the invention provides a molecule comprisingor consisting of the antisense nucleotide sequence of SEQ ID NO 8 to SEQID NO 69. In an even more preferred embodiment, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 16 and/or SEQ ID NO 65. In a most preferred embodiment, theinvention provides a molecule comprising or consisting of the antisensenucleotide sequence of SEQ ID NO 65. It was found that this molecule isvery efficient in modulating splicing of exon 43 of the DMD pre-mRNA ina muscle cell and/or in a patient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 3:5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ which is present in exon 46 of theDMD gene. More preferably, the invention provides a molecule comprisingor consisting of the antisense nucleotide sequence of SEQ ID NO 70 toSEQ ID NO 122. In an even more preferred embodiment, the inventionprovides a molecule comprising or consisting of the antisense nucleotidesequence of SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID NO117. In a most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 117. It was found that this molecule is very efficient in modulatingsplicing of exon 46 of the DMD pre-mRNA in a muscle cell or in apatient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 4: 5′-GGCGGTAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3′ which is present inexon 50 of the DMD gene. More preferably, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535.In an even more preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167. In amost preferred embodiment, the invention provides a molecule comprisingor consisting of the antisense nucleotide sequence of SEQ ID NO 127. Itwas found that this molecule is very efficient in modulating splicing ofexon 50 of the DMD pre-mRNA in a muscle cell and/or in a patient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 5:5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′ which is present in exon 51of the DMD gene. More preferably, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 168 to SEQ ID NO 241.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 6:5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ which is present inexon 52 of the DMD gene. More preferably, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred embodiment,the invention provides a molecule comprising or consisting of theantisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299. Ina most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 299. It was found that this molecule is very efficient in modulatingsplicing of exon 52 of the DMD pre-mRNA in a muscle cell and/or in apatient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 7:5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ which is present in exon 53 of the DMDgene. More preferably, the invention provides a molecule comprising orconsisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQID NO 358. In a most preferred embodiment, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 357. It was found that this molecule is very efficient inmodulating splicing of exon 53 of the DMD pre-mRNA in a muscle celland/or in a patient.

A nucleotide sequence of a molecule of the invention may contain RNAresidues, or one or more DNA residues, and/or one or more nucleotideanalogues or equivalents, as will be further detailed herein below.

It is preferred that a molecule of the invention comprises one or moreresidues that are modified to increase nuclease resistance, and/or toincrease the affinity of the antisense nucleotide for the targetsequence. Therefore, in a preferred embodiment, the antisense nucleotidesequence comprises at least one nucleotide analogue or equivalent,wherein a nucleotide analogue or equivalent is defined as a residuehaving a modified base, and/or a modified backbone, and/or a non-naturalinternucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalentcomprises a modified backbone. Examples of such backbones are providedby morpholino backbones, carbamate backbones, siloxane backbones,sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetylbackbones, methyleneformacetyl backbones, riboacetyl backbones, alkenecontaining backbones, sulfamate, sulfonate and sulfonamide backbones,methyleneimino and methylenehydrazino backbones, and amide backbones.Phosphorodiamidate morpholino oligomers are modified backboneoligonucleotides that have previously been investigated as antisenseagents. Morpholino oligonucleotides have an uncharged backbone in whichthe deoxyribose sugar of DNA is replaced by a six membered ring and thephosphodiester linkage is replaced by a phosphorodiamidate linkage.Morpholino oligonucleotides are resistant to enzymatic degradation andappear to function as antisense agents by arresting translation orinterfering with pre-mRNA splicing rather than by activating RNase H.Morpholino oligonucleotides have been successfully delivered to tissueculture cells by methods that physically disrupt the cell membrane, andone study comparing several of these methods found that scrape loadingwas the most efficient method of delivery; however, because themorpholino backbone is uncharged, cationic lipids are not effectivemediators of morpholino oligonucleotide uptake in cells. A recent reportdemonstrated triplex formation by a morpholino oligonucleotide and,because of the non-ionic backbone, these studies showed that themorpholino oligonucleotide was capable of triplex formation in theabsence of magnesium.

It is further preferred that that the linkage between the residues in abackbone do not include a phosphorus atom, such as a linkage that isformed by short chain alkyl or cycloalkyl internucleoside linkages,mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, orone or more short chain heteroatomic or heterocyclic internucleosidelinkages.

A preferred nucleotide analogue or equivalent comprises a PeptideNucleic Acid (PNA), having a modified polyamide backbone (Nielsen, etal. (1991) Science 254, 1497-1500). PNA-based molecules are true mimicsof DNA molecules in terms of base-pair recognition. The backbone of thePNA is composed of N-(2-aminoethyl)-glycine units linked by peptidebonds, wherein the nucleobases are linked to the backbone by methylenecarbonyl bonds. An alternative backbone comprises a one-carbon extendedpyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun,495-497). Since the backbone of a PNA molecule contains no chargedphosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNAor RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365,566-568).

A further preferred backbone comprises a morpholino nucleotide analog orequivalent, in which the ribose or deoxyribose sugar is replaced by a6-membered morpholino ring. A most preferred nucleotide analog orequivalent comprises a phosphorodiamidate morpholino oligomer (PMO), inwhich the ribose or deoxyribose sugar is replaced by a 6-memberedmorpholino ring, and the anionic phosphodiester linkage between adjacentmorpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent of theinvention comprises a substitution of one of the non-bridging oxygens inthe phosphodiester linkage. This modification slightly destabilizesbase-pairing but adds significant resistance to nuclease degradation. Apreferred nucleotide analogue or equivalent comprises phosphorothioate,chiral phosphorothioate, phosphorodithioate, phosphotriester,aminoalkylphosphotriester, H-phosphonate, methyl and other alkylphosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonateand chiral phosphonate, phosphinate, phosphoramidate including 3′-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate,thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate orboranophosphate.

A further preferred nucleotide analogue or equivalent of the inventioncomprises one or more sugar moieties that are mono- or disubstituted atthe 2′, 3′ and/or 5′ position such as a —OH; —F; substituted orunsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl,alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted byone or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy,-aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and-dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose orderivative thereof, or a deoxypyranose or derivative thereof, preferablya ribose or a derivative thereof, or a deoxyribose or a derivativethereof. Such preferred derivatized sugar moieties comprise LockedNucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or4′ carbon atom of the sugar ring thereby forming a bicyclic sugarmoiety. A preferred LNA comprises 2′-0,4′-C-ethylene-bridged nucleicacid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242).These substitutions render the nucleotide analogue or equivalent RNase Hand nuclease resistant and increase the affinity for the target RNA.

It is understood by a skilled person that it is not necessary for allpositions in an antisense oligonucleotide to be modified uniformly. Inaddition, more than one of the aforementioned analogues or equivalentsmay be incorporated in a single antisense oligonucleotide or even at asingle position within an antisense oligonucleotide. In certainembodiments, an antisense oligonucleotide of the invention has at leasttwo different types of analogues or equivalents.

A preferred antisense oligonucleotide according to the inventioncomprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, suchas 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose,2′-O-propyl modified ribose, and/or substituted derivatives of thesemodifications such as halogenated derivatives.

A most preferred antisense oligonucleotide according to the inventioncomprises of 2′-O-methyl phosphorothioate ribose.

A functional equivalent of a molecule of the invention may be defined asan oligonucleotide as defined herein wherein an activity of saidfunctional equivalent is retained to at least some extent. Preferably,an activity of said functional equivalent is inducing exon 43, 46, 50,51, 52, or 53 skipping and providing a functional dystrophin protein.Said activity of said functional equivalent is therefore preferablyassessed by detection of exon 43, 46, 50, 51, 52, or 53 skipping and byquantifying the amount of functional dystrophin protein. A functionaldystrophin is herein preferably defined as being a dystrophin able tobind actin and members of the DGC protein complex. The assessment ofsaid activity of an oligonucleotide is preferably done by RT-PCR or byimmunofluorescence or Western blot analyses. Said activity is preferablyretained to at least some extent when it represents at least 50%, or atleast 60%, or at least 70% or at least 80% or at least 90% or at least95% or more of corresponding activity of said oligonucleotide thefunctional equivalent derives from. Throughout this application, whenthe word oligonucleotide is used it may be replaced by a functionalequivalent thereof as defined herein.

It will be understood by a skilled person that distinct antisenseoligonucleotides can be combined for efficiently skipping any of exon43, exon 46, exon 50, exon 51, exon 52 and/or exon 53 of the human DMDpre-mRNA. It is encompassed by the present invention to use one, two,three, four, five or more oligonucleotides for skipping one of saidexons (i.e. exon, 43, 46, 50, 51, 52, or 53). It is also encompassed touse at least two oligonucleotides for skipping at least two, of saidexons. Preferably two of said exons are skipped. More preferably, thesetwo exons are:

−43 and 51, or

−43 and 53, or

−50 and 51, or

−51 and 52, or

−52 and 53.

The skilled person will know which combination of exons is preferred tobe skipped depending on the type, the number and the location of themutation present in a DMD or BMD patient.

An antisense oligonucleotide can be linked to a moiety that enhancesuptake of the antisense oligonucleotide in cells, preferably musclecells. Examples of such moieties are cholesterols, carbohydrates,vitamins, biotin, lipids, phospholipids, cell-penetrating peptidesincluding but not limited to antennapedia, TAT, transportan andpositively charged amino acids such as oligoarginine, poly-arginine,oligolysine or polylysine, antigen-binding domains such as provided byan antibody, a Fab fragment of an antibody, or a single chain antigenbinding domain such as a cameloid single domain antigen-binding domain.

A preferred antisense oligonucleotide comprises a peptide-linked PMO.

A preferred antisense oligonucleotide comprising one or more nucleotideanalogs or equivalents of the invention modulates splicing in one ormore muscle cells, including heart muscle cells, upon systemic delivery.In this respect, systemic delivery of an antisense oligonucleotidecomprising a specific nucleotide analog or equivalent might result intargeting a subset of muscle cells, while an antisense oligonucleotidecomprising a distinct nucleotide analog or equivalent might result intargeting of a different subset of muscle cells. Therefore, in oneembodiment it is preferred to use a combination of antisenseoligonucleotides comprising different nucleotide analogs or equivalentsfor inducing skipping of exon 43, 46, 50, 51, 52, or 53 of the human DMDpre-mRNA.

A cell can be provided with a molecule capable of interfering withessential sequences that result in highly efficient skipping of exon 43,exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNAby plasmid-derived antisense oligonucleotide expression or viralexpression provided by adenovirus- or adeno-associated virus-basedvectors. In a preferred embodiment, there is provided a viral-basedexpression vector comprising an expression cassette that drivesexpression of a molecule as identified herein. Expression is preferablydriven by a polymerase III promoter, such as a U1, a U6, or a U7 RNApromoter. A muscle or myogenic cell can be provided with a plasmid forantisense oligonucleotide expression by providing the plasmid in anaqueous solution. Alternatively, a plasmid can be provided bytransfection using known transfection agentia such as, for example,LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500(MBI Fermentas)), or derivatives thereof.

One preferred antisense oligonucleotide expression system is anadenovirus associated virus (AAV)-based vector. Single chain and doublechain AAV-based vectors have been developed that can be used forprolonged expression of small antisense nucleotide sequences for highlyefficient skipping of exon 43, 46, 50, 51, 52 or 53 of the DMD pre-mRNA.

A preferred AAV-based vector comprises an expression cassette that isdriven by a polymerase III-promoter (Pol III). A preferred Pol IIIpromoter is, for example, a U1, a U6, or a U7 RNA promoter.

The invention therefore also provides a viral-based vector, comprising aPol III-promoter driven expression cassette for expression of one ormore antisense sequences of the invention for inducing skipping of exon43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMDpre-mRNA.

Pharmaceutical Composition

If required, a molecule or a vector expressing an antisenseoligonucleotide of the invention can be incorporated into apharmaceutically active mixture or composition by adding apharmaceutically acceptable carrier.

Therefore, in a further aspect, the invention provides a composition,preferably a pharmaceutical composition comprising a molecule comprisingan antisense oligonucleotide according to the invention, and/or aviral-based vector expressing the antisense sequence(s) according to theinvention and a pharmaceutically acceptable carrier.

A preferred pharmaceutical composition comprises a molecule as definedherein and/or a vector as defined herein, and a pharmaceuticalacceptable carrier or excipient, optionally combined with a moleculeand/or a vector as defined herein which is able to induce skipping ofexon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62,63, 65, 66, 69, or 75 of the DMD pre-mRNA. Preferred molecules able toinduce skipping of any of these exon are identified in any one of Tables1 to 7.

Preferred excipients include excipients capable of forming complexes,vesicles and/or liposomes that deliver such a molecule as definedherein, preferably an oligonucleotide complexed or trapped in a vesicleor liposome through a cell membrane. Many of these excipients are knownin the art. Suitable excipients comprise polyethylenimine andderivatives, or similar cationic polymers, including polypropyleneimineor polyethylenimine copolymers (PECs) and derivatives, ExGen 500,synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsidproteins that are capable of self assembly into particles that candeliver such molecule, preferably an oligonucleotide as defined hereinto a cell, preferably a muscle cell. Such excipients have been shown toefficiently deliver (oligonucleotide such as antisense) nucleic acids toa wide variety of cultured cells, including muscle cells. Their hightransfection potential is combined with an excepted low to moderatetoxicity in terms of overall cell survival. The ease of structuralmodification can be used to allow further modifications and the analysisof their further (in vivo) nucleic acid transfer characteristics andtoxicity.

Lipofectin represents an example of a liposomal transfection agent. Itconsists of two lipid components, a cationic lipid N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAPwhich is the methylsulfate salt) and a neutral lipiddioleoylphosphatidylethanolamine (DOPE). The neutral component mediatesthe intracellular release. Another group of delivery systems arepolymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, whichare well known as DNA transfection reagent can be combined withbutylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulatecationic nanoparticles that can deliver a molecule or a compound asdefined herein, preferably an oligonucleotide across cell membranes intocells.

In addition to these common nanoparticle materials, the cationic peptideprotamine offers an alternative approach to formulate a compound asdefined herein, preferably an oligonucleotide as colloids. Thiscolloidal nanoparticle system can form so called proticles, which can beprepared by a simple self-assembly process to package and mediateintracellular release of a compound as defined herein, preferably anoligonucleotide. The skilled person may select and adapt any of theabove or other commercially available alternative excipients anddelivery systems to package and deliver a compound as defined herein,preferably an oligonucleotide for use in the current invention todeliver said compound for the treatment of Duchenne Muscular Dystrophyor Becker Muscular Dystrophy in humans.

In addition, a compound as defined herein, preferably an oligonucleotidecould be covalently or non-covalently linked to a targeting ligandspecifically designed to facilitate the uptake in to the cell, cytoplasmand/or its nucleus. Such ligand could comprise (i) a compound (includingbut not limited to peptide(-like) structures) recognising cell, tissueor organ specific elements facilitating cellular uptake and/or (ii) achemical compound able to facilitate the uptake in to cells and/or theintracellular release of an a compound as defined herein, preferably anoligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, a compound as defined herein,preferably an oligonucleotide are formulated in a medicament which isprovided with at least an excipient and/or a targeting ligand fordelivery and/or a delivery device of said compound to a cell and/orenhancing its intracellular delivery. Accordingly, the invention alsoencompasses a pharmaceutically acceptable composition comprising acompound as defined herein, preferably an oligonucleotide and furthercomprising at least one excipient and/or a targeting ligand for deliveryand/or a delivery device of said compound to a cell and/or enhancing itsintracellular delivery. It is to be understood that a molecule orcompound or oligonucleotide may not be formulated in one singlecomposition or preparation. Depending on their identity, the skilledperson will know which type of formulation is the most appropriate foreach compound.

In a preferred embodiment, an in vitro concentration of a molecule or anoligonucleotide as defined herein, which is ranged between 0.1 nM and 1μM is used. More preferably, the concentration used is ranged between0.3 to 400 nM, even more preferably between 1 to 200 nM. A molecule oran oligonucleotide as defined herein may be used at a dose which isranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If severalmolecules or oligonucleotides are used, these concentrations may referto the total concentration of oligonucleotides or the concentration ofeach oligonucleotide added. The ranges of concentration ofoligonucleotide(s) as given above are preferred concentrations for invitro or ex vivo uses. The skilled person will understand that dependingon the oligonucleotide(s) used, the target cell to be treated, the genetarget and its expression levels, the medium used and the transfectionand incubation conditions, the concentration of oligonucleotide(s) usedmay further vary and may need to be optimised any further.

More preferably, a compound preferably an oligonucleotide to be used inthe invention to prevent, treat DMD or BMD are synthetically producedand administered directly to a cell, a tissue, an organ and/or patientsin formulated form in a pharmaceutically acceptable composition orpreparation. The delivery of a pharmaceutical composition to the subjectis preferably carried out by one or more parenteral injections, e.g.intravenous and/or subcutaneous and/or intramuscular and/or intrathecaland/or intraventricular administrations, preferably injections, at oneor at multiple sites in the human body.

A preferred oligonucleotide as defined herein optionally comprising oneor more nucleotide analogs or equivalents of the invention modulatessplicing in one or more muscle cells, including heart muscle cells, uponsystemic delivery. In this respect, systemic delivery of anoligonucleotide comprising a specific nucleotide analog or equivalentmight result in targeting a subset of muscle cells, while anoligonucleotide comprising a distinct nucleotide analog or equivalentmight result in targeting of a different subset of muscle cells.

In this respect, systemic delivery of an oligonucleotide comprising aspecific nucleotide analog or equivalent might result in targeting asubset of muscle cells, while an oligonucleotide comprising a distinctnucleotide analog or equivalent might result in targeting a differentsubset of muscle cells. Therefore, in this embodiment, it is preferredto use a combination of oligonucleotides comprising different nucleotideanalogs or equivalents for modulating splicing of the DMD mRNA in atleast one type of muscle cells.

In a preferred embodiment, there is provided a molecule or a viral-basedvector for use as a medicament, preferably for modulating splicing ofthe DMD pre-mRNA, more preferably for promoting or inducing skipping ofany of exon 43, 46, 50-53 as identified herein.

Use

In yet a further aspect, the invention provides the use of an antisenseoligonucleotide or molecule according to the invention, and/or aviral-based vector that expresses one or more antisense sequencesaccording to the invention and/or a pharmaceutical composition, formodulating splicing of the DMD pre-mRNA. The splicing is preferablymodulated in a human myogenic cell or muscle cell in vitro. Morepreferred is that splicing is modulated in a human muscle cell in vivo.Accordingly, the invention further relates to the use of the molecule asdefined herein and/or the vector as defined herein and/or or thepharmaceutical composition as defined herein for modulating splicing ofthe DMD pre-mRNA or for the preparation of a medicament for thetreatment of a DMD or BMD patient.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a molecule or a viral-basedvector or a composition as defined herein may comprise additionalcomponent(s) than the ones specifically identified, said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the element ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”. Each embodiment as identified herein may becombined together unless otherwise indicated. All patent and literaturereferences cited in the present specification are hereby incorporated byreference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

EXAMPLES Examples 1-4

Materials and Methods

AON design was based on (partly) overlapping open secondary structuresof the target exon RNA as predicted by the m-fold program, on (partly)overlapping putative SR-protein binding sites as predicted by theESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V.(Leiden, Netherlands), and contain 2′-O-methyl RNA and full-lengthphosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

Myotube cultures derived from a healthy individual (“human control”)(examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patientcarrying an exon 45 deletion (example 2; exon 46 skipping) wereprocessed as described previously (Aartsma-Rus et al., Neuromuscul.Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For thescreening of AONs, myotube cultures were transfected with 50 nM and 150nM (example 2), 200 nM and 500 nM (example 4) or 500 nM only (examples 1and 3) of each AON. Transfection reagent UNIFectylin (ProsensaTherapeutics BV, Netherlands) was used, with 2 μl UNIFectylin perm AON.Exon skipping efficiencies were determined by nested RT-PCR analysisusing primers in the exons flanking the targeted exons (43, 46, 50, 51,52, or 53). PCR fragments were isolated from agarose gels for sequenceverification. For quantification, the PCR products were analyzed usingthe DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (AgilentTechnologies, USA).

Results

DMD Exon 43 Skipping.

A series of AONs targeting sequences within exon 43 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 43 herein definedas SEQ ID NO 2, was indeed capable of inducing exon 43 skipping. PS237(SEQ ID NO: 65) reproducibly induced highest levels of exon 43 skipping(up to 66%) at 500 nM, as shown in FIG. 1. For comparison, also PS238and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36%respectively (FIG. 1). The precise skipping of exon 43 was confirmed bysequence analysis of the novel smaller transcript fragments. No exon 43skipping was observed in non-treated cells (NT).

DMD Exon 46 Skipping.

A series of AONs targeting sequences within exon 46 were designed andtransfected in myotube cultures derived from a DMD patient carrying anexon 45 deletion in the DMD gene. For patients with such mutationantisense-induced exon 46 skipping would induce the synthesis of anovel, BMD-like dystrophin protein that may indeed alleviate one or moresymptoms of the disease. Subsequent RT-PCR and sequence analysis ofisolated RNA demonstrated that almost all AONs targeting a continuousnucleotide stretch within exon 46 herein defined as SEQ ID NO 3, wasindeed capable of inducing exon 46 skipping, even at relatively low AONconcentrations of 50 nM. PS182 (SEQ ID NO: 117) reproducibly inducedhighest levels of exon 46 skipping (up to 50% at 50 nM and 74% at 150nM), as shown in FIG. 2. For comparison, also PS177, PS179, and PS181are shown, inducing exon 46 skipping levels up to 55%, 58% and 42%respectively at 150 nM (FIG. 2). The precise skipping of exon 46 wasconfirmed by sequence analysis of the novel smaller transcriptfragments. No exon 46 skipping was observed in non-treated cells (NT).

DMD Exon 50 Skipping.

A series of AONs targeting sequences within exon 50 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 50 herein definedas SEQ ID NO 4, was indeed capable of inducing exon 50 skipping. PS248(SEQ ID NO: 127) reproducibly induced highest levels of exon 50 skipping(up to 35% at 500 nM), as shown in FIG. 3. For comparison, also PS245,PS246, and PS247 are shown, inducing exon 50 skipping levels up to14-16% at 500 nM (FIG. 3). The precise skipping of exon 50 was confirmedby sequence analysis of the novel smaller transcript fragments. No exon50 skipping was observed in non-treated cells (NT).

DMD Exon 51 Skipping.

A series of AONs targeting sequences within exon 51 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 51 herein definedas SEQ ID NO 5, was indeed capable of inducing exon 51 skipping. The AONwith SEQ ID NO 180 reproducibly induced highest levels of exon 51skipping (not shown).

DMD Exon 52 Skipping.

A series of AONs targeting sequences within exon 52 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 52 herein definedas SEQ ID NO 6, was indeed capable of inducing exon 52 skipping. PS236(SEQ ID NO: 299) reproducibly induced highest levels of exon 52 skipping(up to 88% at 200 nM and 91% at 500 nM), as shown in FIG. 4. Forcomparison, also PS232 and AON 52-1 (previously published by Aartsma-Ruset al. Oligonucleotides 2005) are shown, inducing exon 52 skipping atlevels up to 59% and 10% respectively when applied at 500 nM (FIG. 4).The precise skipping of exon 52 was confirmed by sequence analysis ofthe novel smaller transcript fragments. No exon 52 skipping was observedin non-treated cells (NT).

DMD Exon 53 Skipping.

A series of AONs targeting sequences within exon 53 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 53 herein definedas SEQ ID NO 7, was indeed capable of inducing exon 53 skipping. The AONwith SEQ ID NO 328 reproducibly induced highest levels of exon 53skipping (not shown).

TABLE 1 oligonucleotides for skipping DMD Gene Exon 43 SEQ IDCCACAGGCGUUGCACUUUGCAAUGC SEQ ID NO 39 UCUUCUUGCUAUGAAUAAUGUCAAU NO 8SEQ ID CACAGGCGUUGCACUUUGCAAUGCU SEQ ID NO 40 CUUCUUGCUAUGAAUAAUGUCAAUCNO 9 SEQ ID ACAGGCGUUGCACUUUGCAAUGCUG SEQ ID NO 41UUCUUGCUAUGAAUAAUGUCAAUCC NO 10 SEQ ID CAGGCGUUGCACUUUGCAAUGCUGCSEQ ID NO 42 UCUUGCUAUGAAUAAUGUCAAUCCG NO 11 SEQ IDAGGCGUUGCACUUUGCAAUGCUGCU SEQ ID NO 43 CUUGCUAUGAAUAAUGUCAAUCCGA NO 12SEQ ID GGCGUUGCACUUUGCAAUGCUGCUG SEQ ID NO 44 UUGCUAUGAAUAAUGUCAAUCCGACNO 13 SEQ ID GCGUUGCACUUUGCAAUGCUGCUGU SEQ ID NO 45UGCUAUGAAUAAUGUCAAUCCGACC NO 14 SEQ ID CGUUGCACUUUGCAAUGCUGCUGUCSEQ ID NO 46 GCUAUGAAUAAUGUCAAUCCGACCU NO 15 SEQ IDCGUUGCACUUUGCAAUGCUGCUG SEQ ID NO 47 CUAUGAAUAAUGUCAAUCCGACCUG NO 16PS240 SEQ ID GUUGCACUUUGCAAUGCUGCUGUCU SEQ ID NO 48UAUGAAUAAUGUCAAUCCGACCUGA NO 17 SEQ ID UUGCACUUUGCAAUGCUGCUGUCUUSEQ ID NO 49 AUGAAUAAUGUCAAUCCGACCUGAG NO 18 SEQ IDUGCACUUUGCAAUGCUGCUGUCUUC SEQ ID NO 50 UGAAUAAUGUCAAUCCGACCUGAGC NO 19SEQ ID GCACUUUGCAAUGCUGCUGUCUUCU SEQ ID NO 51 GAAUAAUGUCAAUCCGACCUGAGCUNO 20 SEQ ID CACUUUGCAAUGCUGCUGUCUUCUU SEQ ID NO 52AAUAAUGUCAAUCCGACCUGAGCUU NO 21 SEQ ID ACUUUGCAAUGCUGCUGUCUUCUUGSEQ ID NO 53 AUAAUGUCAAUCCGACCUGAGCUUU NO 22 SEQ IDCUUUGCAAUGCUGCUGUCUUCUUGC SEQ ID NO 54 UAAUGUCAAUCCGACCUGAGCUUUG NO 23SEQ ID UUUGCAAUGCUGCUGUCUUCUUGCU SEQ ID NO 55 AAUGUCAAUCCGACCUGAGCUUUGUNO 24 SEQ ID UUGCAAUGCUGCUGUCUUCUUGCUA SEQ ID NO 56AUGUCAAUCCGACCUGAGCUUUGUU NO 25 SEQ ID UGCAAUGCUGCUGUCUUCUUGCUAUSEQ ID NO 57 UGUCAAUCCGACCUGAGCUUUGUUG NO 26 SEQ IDGCAAUGCUGCUGUCUUCUUGCUAUG SEQ ID NO 58 GUCAAUCCGACCUGAGCUUUGUUGU NO 27SEQ ID CAAUGCUGCUGUCUUCUUGCUAUGA SEQ ID NO 59 UCAAUCCGACCUGAGCUUUGUUGUANO 28 SEQ ID AAUGCUGCUGUCUUCUUGCUAUGAA SEQ ID NO 60CAAUCCGACCUGAGCUUUGUUGUAG NO 29 SEQ ID AUGCUGCUGUCUUCUUGCUAUGAAUSEQ ID NO 61 AAUCCGACCUGAGCUUUGUUGUAGA NO 30 SEQ IDUGCUGCUGUCUUCUUGCUAUGAAUA SEQ ID NO 62 AUCCGACCUGAGCUUUGUUGUAGAC NO 31SEQ ID GCUGCUGUCUUCUUGCUAUGAAUAA SEQ ID NO 63 UCCGACCUGAGCUUUGUUGUAGACUNO 32 SEQ ID CUGCUGUCUUCUUGCUAUGAAUAAU SEQ ID NO 64CCGACCUGAGCUUUGUUGUAGACUA NO 33 SEQ ID UGCUGUCUUCUUGCUAUGAAUAAUSEQ ID NO 65 CGACCUGAGCUUUGUUGUAG NO 34 G PS237 SEQ IDGCUGUCUUCUUGCUAUGAAUAAUG SEQ ID NO 66 CGACCUGAGCUUUGUUGUAGACUAU NO 35 UPS238 SEQ ID CUGUCUUCUUGCUAUGAAUAAUGUC SEQ ID NO 67GACCUGAGCUUUGUUGUAGACUAUC NO 36 SEQ ID UGUCUUCUUGCUAUGAAUAAUGUCSEQ ID NO 68 ACCUGAGCUUUGUUGUAGACUAUCA NO 37 A SEQ IDGUCUUCUUGCUAUGAAUAAUGUCA SEQ ID NO 69 CCUGA GCUUU GUUGU AGACU AUC NO 38A

TABLE 2 oligonucleotides for skipping DMD Gene Exon 46 SEQ IDGCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID NO 97 CCAGGUUCAAGUGGGAUACUAGCAA NO 70PS179 SEQ ID CUUUUCUUUUAGUUGCUGCUCUUUU SEQ ID NO 98CAGGUUCAAGUGGGAUACUAGCAAU NO 71 SEQ ID UUUUCUUUUAGUUGCUGCUCUUUUCSEQ ID NO 99 AGGUUCAAGUGGGAUACUAGCAAUG NO 72 SEQ IDUUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO GGUUCAAGUGGGAUACUAGCAAUGU NO 73 100SEQ ID UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID NO GUUCAAGUGGGAUACUAGCAAUGUUNO 74 101 SEQ ID UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NOUUCAAGUGGGAUACUAGCAAUGUUA NO 75 102 SEQ ID CUUUUAGUUGCUGCUCUUUUCCAGGSEQ ID NO UCAAGUGGGAUACUAGCAAUGUUAU NO 76 103 SEQ IDUUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID NO CAAGUGGGAUACUAGCAAUGUUAUC NO 77 104SEQ ID UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO AAGUGGGAUACUAGCAAUGUUAUCUNO 78 105 SEQ ID UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NOAGUGGGAUACUAGCAAUGUUAUCUG NO 79 106 SEQ ID UAGUUGCUGCUCUUUUCCAGGUUCASEQ ID NO GUGGGAUACUAGCAAUGUUAUCUGC NO 80 107 SEQ IDAGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO UGGGAUACUAGCAAUGUUAUCUGCU NO 81 108SEQ ID GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO GGGAUACUAGCAAUGUUAUCUGCUUNO 82 109 SEQ ID UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID NOGGAUACUAGCAAUGUUAUCUGCUUC NO 83 110 PS181 SEQ IDUGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO GAUACUAGCAAUGUUAUCUGCUUCC NO 84 111SEQ ID GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO AUACUAGCAAUGUUAUCUGCUUCCUNO 85 112 SEQ ID CUGCUCUUUUCCAGGUUCAAGUGGG SEQ ID NOUACUAGCAAUGUUAUCUGCUUCCUC NO 86 113 SEQ ID UGCUCUUUUCCAGGUUCAAGUGGGASEQ ID NO ACUAGCAAUGUUAUCUGCUUCCUCC NO 87 114 SEQ IDGCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO CUAGCAAUGUUAUCUGCUUCCUCCA NO 88 115SEQ ID CUCUUUUCCAGGUUCAAGUGGGAUA SEQ ID NO UAGCAAUGUUAUCUGCUUCCUCCAANO 89 116 SEQ ID UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NOAGCAAUGUUAUCUGCUUCCUCCAAC NO 90 117 PS182 SEQ ID UCUUUUCCAGGUUCAAGUGGSEQ ID NO GCAAUGUUAUCUGCUUCCUCCAACC NO 91 118 PS177 SEQ IDCUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO CAAUGUUAUCUGCUUCCUCCAACCA NO 92 119SEQ ID UUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO AAUGUUAUCUGCUUCCUCCAACCAUNO 93 A 120 SEQ ID UUUCCAGGUUCAAGUGGGAUACUA SEQ ID NOAUGUUAUCUGCUUCCUCCAACCAUA NO 94 G 121 SEQ ID UUCCAGGUUCAAGUGGGAUACUAGCSEQ ID NO UGUUAUCUGCUUCCUCCAACCAUAA NO 95 122 SEQ IDUCCAGGUUCAAGUGGGAUACUAGCA NO 96

TABLE 3 oligonucleotides for skipping DMD Gene Exon 50 SEQ IDCCAAUAGUGGUCAGUCCAGGAGCUA SEQ ID NO CUAGGUCAGGCUGCUUUGCCCUCAG NO 123 146SEQ ID CAAUAGUGGUCAGUCCAGGAGCUAG SEQ ID NO UAGGUCAGGCUGCUUUGCCCUCAGCNO 124 147 SEQ ID AAUAGUGGUCAGUCCAGGAGCUAGG SEQ ID NOAGGUCAGGCUGCUUUGCCCUCAGCU NO 125 148 SEQ ID AUAGUGGUCAGUCCAGGAGCUAGGUSEQ ID NO GGUCAGGCUGCUUUGCCCUCAGCUC NO 126 149 SEQ IDAUAGUGGUCAGUCCAGGAGCU SEQ ID NO GUCAGGCUGCUUUGCCCUCAGCUCU NO 127 150PS248 SEQ ID UAGUGGUCAGUCCAGGAGCUAGGUC SEQ ID NOUCAGGCUGCUUUGCCCUCAGCUCUU NO 128 151 SEQ ID AGUGGUCAGUCCAGGAGCUAGGUCASEQ ID NO CAGGCUGCUUUGCCCUCAGCUCUUG NO 129 152 SEQ IDGUGGUCAGUCCAGGAGCUAGGUCAG SEQ ID NO AGGCUGCUUUGCCCUCAGCUCUUGA NO 130 153SEQ ID UGGUCAGUCCAGGAGCUAGGUCAGG SEQ ID NO GGCUGCUUUGCCCUCAGCUCUUGAANO 131 154 SEQ ID GGUCAGUCCAGGAGCUAGGUCAGGC SEQ ID NOGCUGCUUUGCCCUCAGCUCUUGAAG NO 132 155 SEQ ID GUCAGUCCAGGAGCUAGGUCAGGCUSEQ ID NO CUGCUUUGCCCUCAGCUCUUGAAGU NO 133 156 SEQ IDUCAGUCCAGGAGCUAGGUCAGGCUG SEQ ID NO UGCUUUGCCCUCAGCUCUUGAAGUA NO 134 157SEQ ID CAGUCCAGGAGCUAGGUCAGGCUGC SEQ ID NO GCUUUGCCCUCAGCUCUUGAAGUAANO 135 158 SEQ ID AGUCCAGGAGCUAGGUCAGGCUGCU SEQ ID NOCUUUGCCCUCAGCUCUUGAAGUAAA NO 136 159 SEQ ID GUCCAGGAGCUAGGUCAGGCUGCUUSEQ ID NO UUUGCCCUCAGCUCUUGAAGUAAAC NO 137 160 SEQ IDUCCAGGAGCUAGGUCAGGCUGCUUU SEQ ID NO UUGCCCUCAGCUCUUGAAGUAAACG NO 138 161SEQ ID CCAGGAGCUAGGUCAGGCUGCUUUG SEQ ID NO UGCCCUCAGCUCUUGAAGUAAACGGNO 139 162 SEQ ID CAGGAGCUAGGUCAGGCUGCUUUGC SEQ ID NOGCCCUCAGCUCUUGAAGUAAACGGU NO 140 163 SEQ ID AGGAGCUAGGUCAGGCUGCUUUGCCSEQ ID NO CCCUCAGCUCUUGAAGUAAACGGUU NO 141 164 SEQ IDGGAGCUAGGUCAGGCUGCUUUGCCC SEQ ID NO CCUCAGCUCUUGAAGUAAAC NO 142 165PS246 SEQ ID GAGCUAGGUCAGGCUGCUUUGCCCU SEQ ID NO CCUCAGCUCUUGAAGUAAACGNO 143 166 PS247 SEQ ID AGCUAGGUCAGGCUGCUUUGCCCUC SEQ ID NOCUCAGCUCUUGAAGUAAACG NO 144 167 PS245 SEQ ID GCUAGGUCAGGCUGCUUUGCCCUCASEQ ID NO CCUCAGCUCUUGAAGUAAACGGUUU NO 145 529 SEQ IDCUCAGCUCUUGAAGUAAACGGUUUA SEQ ID NO UCAGCUCUUGAAGUAAACGGUUUAC NO 530 531SEQ ID CAGCUCUUGAAGUAAACGGUUUACC SEQ ID NO AGCUCUUGAAGUAAACGGUUUACCGNO 532 533 SEQ ID GCUCUUGAAGUAAACGGUUUACCGC SEQ ID NOCUCUUGAAGUAAACGGUUUACCGCC NO 534 535

TABLE 4 oligonucleotides for skipping DMD Gene Exon 51 SEQ IDGUACCUCCAACAUCAAGGAAGAUGG SEQ ID NO GAGAUGGCAGUUUCCUUAGUAACCA NO 168 205SEQ ID UACCUCCAACAUCAAGGAAGAUGGC SEQ ID NO AGAUGGCAGUUUCCUUAGUAACCACNO 169 206 SEQ ID ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NOGAUGGCAGUUUCCUUAGUAACCACA NO 170 207 SEQ ID CCUCCAACAUCAAGGAAGAUGGCAUSEQ ID NO AUGGCAGUUUCCUUAGUAACCACAG NO 171 208 SEQ IDCUCCAACAUCAAGGAAGAUGGCAUU SEQ ID NO UGGCAGUUUCCUUAGUAACCACAGG NO 172 209SEQ ID UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID NO GGCAGUUUCCUUAGUAACCACAGGUNO 173 210 SEQ ID CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID NOGCAGUUUCCUUAGUAACCACAGGUU NO 174 211 SEQ ID CAACAUCAAGGAAGAUGGCAUUUCUSEQ ID NO CAGUUUCCUUAGUAACCACAGGUUG NO 175 212 SEQ IDAACAUCAAGGAAGAUGGCAUUUCUA SEQ ID NO AGUUUCCUUAGUAACCACAGGUUGU NO 176 213SEQ ID ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO GUUUCCUUAGUAACCACAGGUUGUGNO 177 214 SEQ ID CAUCAAGGAAGAUGGCAUUUCUAGU SEQ ID NOUUUCCUUAGUAACCACAGGUUGUGU NO 178 215 SEQ ID AUCAAGGAAGAUGGCAUUUCUAGUUSEQ ID NO UUCCUUAGUAACCACAGGUUGUGUC NO 179 216 SEQ IDUCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO UCCUUAGUAACCACAGGUUGUGUCA NO 180 217SEQ ID CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID NO CCUUAGUAACCACAGGUUGUGUCACNO 181 218 SEQ ID AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NOCUUAGUAACCACAGGUUGUGUCACC NO 182 219 SEQ ID AGGAAGAUGGCAUUUCUAGUUUGGASEQ ID NO UUAGUAACCACAGGUUGUGUCACCA NO 183 220 SEQ IDGGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID NO UAGUAACCACAGGUUGUGUCACCAG NO 184 221SEQ ID GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID NO AGUAACCACAGGUUGUGUCACCAGANO 185 222 SEQ ID AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NOGUAACCACAGGUUGUGUCACCAGAG NO 186 223 SEQ ID AGAUGGCAUUUCUAGUUUGGAGAUGSEQ ID NO UAACCACAGGUUGUGUCACCAGAGU NO 187 224 SEQ IDGAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID NO AACCACAGGUUGUGUCACCAGAGUA NO 188 225SEQ ID AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID NO ACCACAGGUUGUGUCACCAGAGUAANO 189 226 SEQ ID UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID NOCCACAGGUUGUGUCACCAGAGUAAC NO 190 227 SEQ ID GGCAUUUCUAGUUUGGAGAUGGCAGSEQ ID NO CACAGGUUGUGUCACCAGAGUAACA NO 191 228 SEQ IDGCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO ACAGGUUGUGUCACCAGAGUAACAG NO 192 229SEQ ID CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID NO CAGGUUGUGUCACCAGAGUAACAGUNO 193 230 SEQ ID AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID NOAGGUUGUGUCACCAGAGUAACAGUC NO 194 231 SEQ ID UUUCUAGUUUGGAGAUGGCAGUUUCSEQ ID NO GGUUGUGUCACCAGAGUAACAGUCU NO 195 232 SEQ IDUUCUAGUUUGGAGAUGGCAGUUUCC SEQ ID NO GUUGUGUCACCAGAGUAACAGUCUG NO 196 233SEQ ID UCUAGUUUGGAGAUGGCAGUUUCCU SEQ ID NO UUGUGUCACCAGAGUAACAGUCUGANO 197 234 SEQ ID CUAGUUUGGAGAUGGCAGUUUCCUU SEQ ID NOUGUGUCACCAGAGUAACAGUCUGAG NO 198 235 SEQ ID UAGUUUGGAGAUGGCAGUUUCCUUASEQ ID NO GUGUCACCAGAGUAACAGUCUGAGU NO 199 236 SEQ IDAGUUUGGAGAUGGCAGUUUCCUUAG SEQ ID NO UGUCACCAGAGUAACAGUCUGAGUA NO 200 237SEQ ID GUUUGGAGAUGGCAGUUUCCUUAGU SEQ ID NO GUCACCAGAGUAACAGUCUGAGUAGNO 201 238 SEQ ID UUUGGAGAUGGCAGUUUCCUUAGUA SEQ ID NOUCACCAGAGUAACAGUCUGAGUAGG NO 202 239 SEQ ID UUGGAGAUGGCAGUUUCCUUAGUAASEQ ID NO CACCAGAGUAACAGUCUGAGUAGGA NO 203 240 SEQ IDUGGAGAUGGCAGUUUCCUUAGUAAC SEQ ID NO ACCAGAGUAACAGUCUGAGUAGGAG NO 204 241

TABLE 5 oligonucleotides for skipping DMD Gene Exon 52 SEQ IDAGCCUCUUGAUUGCUGGUCUUGUUU SEQ ID NO UUGGGCAGCGGUAAUGAGUUCUUCC NO 242 277SEQ ID GCCUCUUGAUUGCUGGUCUUGUUUU SEQ ID NO UGGGCAGCGGUAAUGAGUUCUUCCANO 243 278 SEQ ID CCUCUUGAUUGCUGGUCUUGUUUUU SEQ ID NOGGGCAGCGGUAAUGAGUUCUUCCAA NO 244 279 SEQ ID CCUCUUGAUUGCUGGUCUUGSEQ ID NO GGCAGCGGUAAUGAGUUCUUCCAAC NO 245 280 SEQ IDCUCUUGAUUGCUGGUCUUGUUUUUC SEQ ID NO GCAGCGGUAAUGAGUUCUUCCAACU NO 246 281PS232 SEQ ID UCUUGAUUGCUGGUCUUGUUUUUCA SEQ ID NOCAGCGGUAAUGAGUUCUUCCAACUG NO 247 282 SEQ ID CUUGAUUGCUGGUCUUGUUUUUCAASEQ ID NO AGCGGUAAUGAGUUCUUCCAACUGG NO 248 283 SEQ IDUUGAUUGCUGGUCUUGUUUUUCAAA SEQ ID NO GCGGUAAUGAGUUCUUCCAACUGGG NO 249 284SEQ ID UGAUUGCUGGUCUUGUUUUUCAAAU SEQ ID NO CGGUAAUGAGUUCUUCCAACUGGGGNO 250 285 SEQ ID GAUUGCUGGUCUUGUUUUUCAAAUU SEQ ID NOGGUAAUGAGUUCUUCCAACUGGGGA NO 251 286 SEQ ID GAUUGCUGGUCUUGUUUUUCSEQ ID NO GGUAAUGAGUUCUUCCAACUGG NO 252 287 SEQ IDAUUGCUGGUCUUGUUUUUCAAAUUU SEQ ID NO GUAAUGAGUUCUUCCAACUGGGGAC NO 253 288SEQ ID UUGCUGGUCUUGUUUUUCAAAUUUU SEQ ID NO UAAUGAGUUCUUCCAACUGGGGACGNO 254 289 SEQ ID UGCUGGUCUUGUUUUUCAAAUUUUG SEQ ID NOAAUGAGUUCUUCCAACUGGGGACGC NO 255 290 SEQ ID GCUGGUCUUGUUUUUCAAAUUUUGGSEQ ID NO AUGAGUUCUUCCAACUGGGGACGCC NO 256 291 SEQ IDCUGGUCUUGUUUUUCAAAUUUUGGG SEQ ID NO UGAGUUCUUCCAACUGGGGACGCCU NO 257 292SEQ ID UGGUCUUGUUUUUCAAAUUUUGGGC SEQ ID NO GAGUUCUUCCAACUGGGGACGCCUCNO 258 293 SEQ ID GGUCUUGUUUUUCAAAUUUUGGGCA SEQ ID NOAGUUCUUCCAACUGGGGACGCCUCU NO 259 294 SEQ ID GUCUUGUUUUUCAAAUUUUGGGCAGSEQ ID NO GUUCUUCCAACUGGGGACGCCUCUG NO 260 295 SEQ IDUCUUGUUUUUCAAAUUUUGGGCAGC SEQ ID NO UUCUUCCAACUGGGGACGCCUCUGU NO 261 296SEQ ID CUUGUUUUUCAAAUUUUGGGCAGCG SEQ ID NO UCUUCCAACUGGGGACGCCUCUGUUNO 262 297 SEQ ID UUGUUUUUCAAAUUUUGGGCAGCGG SEQ ID NOCUUCCAACUGGGGACGCCUCUGUUC NO 263 298 SEQ ID UGUUUUUCAAAUUUUGGGCAGCGGUSEQ ID NO UUCCAACUGGGGACGCCUCUGUUCC NO 264 299 PS236 SEQ IDGUUUUUCAAAUUUUGGGCAGCGGUA SEQ ID NO UCCAACUGGGGACGCCUCUGUUCCA NO 265 300SEQ ID UUUUUCAAAUUUUGGGCAGCGGUAA SEQ ID NO CCAACUGGGGACGCCUCUGUUCCAANO 266 301 SEQ ID UUUUCAAAUUUUGGGCAGCGGUAAU SEQ ID NOCAACUGGGGACGCCUCUGUUCCAAA NO 267 302 SEQ ID UUUCAAAUUUUGGGCAGCGGUAAUGSEQ ID NO AACUGGGGACGCCUCUGUUCCAAAU NO 268 303 SEQ IDUUCAAAUUUUGGGCAGCGGUAAUGA SEQ ID NO ACUGGGGACGCCUCUGUUCCAAAUC NO 269 304SEQ ID UCAAAUUUUGGGCAGCGGUAAUGAG SEQ ID NO CUGGGGACGCCUCUGUUCCAAAUCCNO 270 305 SEQ ID CAAAUUUUGGGCAGCGGUAAUGAGU SEQ ID NOUGGGGACGCCUCUGUUCCAAAUCCU NO 271 306 SEQ ID AAAUUUUGGGCAGCGGUAAUGAGUUSEQ ID NO GGGGACGCCUCUGUUCCAAAUCCUG NO 272 307 SEQ IDAAUUUUGGGCAGCGGUAAUGAGUUC SEQ ID NO GGGACGCCUCUGUUCCAAAUCCUGC NO 273 308SEQ ID AUUUUGGGCAGCGGUAAUGAGUUCU SEQ ID NO GGACGCCUCUGUUCCAAAUCCUGCANO 274 309 SEQ ID UUUUGGGCAGCGGUAAUGAGUUCUU SEQ ID NOGACGCCUCUGUUCCAAAUCCUGCAU NO 275 310 SEQ ID UUUGGGCAGCGGUAAUGAGUUCUUCNO 276

TABLE 6 oligonucleotides for skipping DMD Gene Exon 53 SEQ IDCUCUGGCCUGUCCUAAGACCUGCUC SEQ ID NO CAGCUUCUUCCUUAGCUUCCAGCCA NO 311 335SEQ ID UCUGGCCUGUCCUAAGACCUGCUCA SEQ ID NO AGCUUCUUCCUUAGCUUCCAGCCAUNO 312 336 SEQ ID CUGGCCUGUCCUAAGACCUGCUCAG SEQ ID NOGCUUCUUCCUUAGCUUCCAGCCAUU NO 313 337 SEQ ID UGGCCUGUCCUAAGACCUGCUCAGCSEQ ID NO CUUCUUCCUUAGCUUCCAGCCAUUG NO 314 338 SEQ IDGGCCUGUCCUAAGACCUGCUCAGCU SEQ ID NO UUCUUCCUUAGCUUCCAGCCAUUGU NO 315 339SEQ ID GCCUGUCCUAAGACCUGCUCAGCUU SEQ ID NO UCUUCCUUAGCUUCCAGCCAUUGUGNO 316 340 SEQ ID CCUGUCCUAAGACCUGCUCAGCUUC SEQ ID NOCUUCCUUAGCUUCCAGCCAUUGUGU NO 317 341 SEQ ID CUGUCCUAAGACCUGCUCAGCUUCUSEQ ID NO UUCCUUAGCUUCCAGCCAUUGUGUU NO 318 342 SEQ IDUGUCCUAAGACCUGCUCAGCUUCUU SEQ ID NO UCCUUAGCUUCCAGCCAUUGUGUUG NO 319 343SEQ ID GUCCUAAGACCUGCUCAGCUUCUUC SEQ ID NO CCUUAGCUUCCAGCCAUUGUGUUGANO 320 344 SEQ ID UCCUAAGACCUGCUCAGCUUCUUCC SEQ ID NOCUUAGCUUCCAGCCAUUGUGUUGAA NO 321 345 SEQ ID CCUAAGACCUGCUCAGCUUCUUCCUSEQ ID NO UUAGCUUCCAGCCAUUGUGUUGAAU NO 322 346 SEQ IDCUAAGACCUGCUCAGCUUCUUCCUU SEQ ID NO UAGCUUCCAGCCAUUGUGUUGAAUC NO 323 347SEQ ID UAAGACCUGCUCAGCUUCUUCCUUA SEQ ID NO AGCUUCCAGCCAUUGUGUUGAAUCCNO 324 348 SEQ ID AAGACCUGCUCAGCUUCUUCCUUAG SEQ ID NOGCUUCCAGCCAUUGUGUUGAAUCCU NO 325 349 SEQ ID AGACCUGCUCAGCUUCUUCCUUAGCSEQ ID NO CUUCCAGCCAUUGUGUUGAAUCCUU NO 326 350 SEQ IDGACCUGCUCAGCUUCUUCCUUAGCU SEQ ID NO UUCCAGCCAUUGUGUUGAAUCCUUU NO 327 351SEQ ID ACCUGCUCAGCUUCUUCCUUAGCUU SEQ ID NO UCCAGCCAUUGUGUUGAAUCCUUUANO 328 352 SEQ ID CCUGCUCAGCUUCUUCCUUAGCUUC SEQ ID NOCCAGCCAUUGUGUUGAAUCCUUUAA NO 329 353 SEQ ID CUGCUCAGCUUCUUCCUUAGCUUCCSEQ ID NO CAGCCAUUGUGUUGAAUCCUUUAAC NO 330 354 SEQ IDUGCUCAGCUUCUUCCUUAGCUUCCA SEQ ID NO AGCCAUUGUGUUGAAUCCUUUAACA NO 331 355SEQ ID GCUCAGCUUCUUCCUUAGCUUCCAG SEQ ID NO GCCAUUGUGUUGAAUCCUUUAACAUNO 332 356 SEQ ID CUCAGCUUCUUCCUUAGCUUCCAGC SEQ ID NOCCAUUGUGUUGAAUCCUUUAACAUU NO 333 357 SEQ ID UCAGCUUCUUCCUUAGCUUCCAGCCSEQ ID NO CAUUGUGUUGAAUCCUUUAACAUUU NO 334 358

TABLE 7oligonucleotides for skipping other exons of the DMD gene as identifiedDMD Gene Exon 6 SEQ ID CAUUUUUGACCUACAUGUGG SEQ ID NOAUUUUUGACCUACAUGGGAAAG NO 359 364 SEQ ID UUUGACCUACAUGUGGAAAG SEQ ID NOUACGAGUUGAUUGUCGGACCCAG NO 360 365 SEQ ID UACAUUUUUGACCUACAUGUGGAASEQ ID NO GUGGUCUCCUUACCUAUGACUGUGG NO 361 A G 366 SEQ IDGGUCUCCUUACCUAUGA SEQ ID NO UGUCUCAGUAAUCUUCUUACCUAU NO 362 367 SEQ IDUCUUACCUAUGACUAUGGAUGAGA NO 363 DMD Gene Exon 7 SEQ IDUGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 370 AUUUACCAACCUUCAGGAUCGAGU NO 368 ASEQ ID CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 371 GGCCUAAAACACAUACACAUANO 369 DMD Gene Exon 11 SEQ ID CCCUGAGGCAUUCCCAUCUUGAAU SEQ IDCUUGAAUUUAGGAGAUUCAUCU NO 372 NO 374 G SEQ ID AGGACUUACUUGCUUUGUUUSEQ ID CAUCUUCUGAUAAUUUUCCUGUU NO 373 NO 375 DMD Gene Exon 17 SEQ IDCCAUUACAGUUGUCUGUGUU SEQ ID UAAUCUGCCUCUUCUUUUGG NO 376 NO 378 SEQ IDUGACAGCCUGUGAAAUCUGUGAG NO 377 DMD Gene Exon 19 SEQ IDCAGCAGUAGUUGUCAUCUGC SEQ ID GCCUGAGCUGAUCUGCUGGCAUC NO 379 NO 381UUGCAGUU SEQ ID GCCUGAGCUGAUCUGCUGGCAUCUUGC SEQ ID UCUGCUGGCAUCUUGCNO 380 NO 382 DMD Gene Exon 21 SEQ ID GCCGGUUGACUUCAUCCUGUGC SEQ IDCUGCAUCCAGGAACAUGGGUCC NO 383 NO 386 SEQ ID GUCUGCAUCCAGGAACAUGGGUCSEQ ID GUUGAAGAUCUGAUAGCCGGUUGA NO 384 NO 387 SEQ IDUACUUACUGUCUGUAGCUCUUUCU NO 385 DMD Gene Exon 44 SEQ IDUCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAA NO 388 NO 413SEQ ID UUCAGCUUCUGUUAGCCACU SEQ ID CAGCUUCUGUUAGCCACUGAUUAAA NO 389NO 414 SEQ ID UUCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAANO 390 NO 415 SEQ ID UCAGCUUCUGUUAGCCACUGA SEQ ID AGCUUCUGUUAGCCACUGAUNO 391 NO 416 SEQ ID UUCAGCUUCUGUUAGCCACUGA SEQ ID GCUUCUGUUAGCCACUGAUUNO 392 NO 417 SEQ ID UCAGCUUCUGUUAGCCACUGA SEQ ID AGCUUCUGUUAGCCACUGAUUNO 393 NO 418 SEQ ID UUCAGCUUCUGUUAGCCACUGA SEQ ID GCUUCUGUUAGCCACUGAUUANO 394 NO 419 SEQ ID UCAGCUUCUGUUAGCCACUGAU SEQ IDAGCUUCUGUUAGCCACUGAUUA NO 395 NO 420 SEQ ID UUCAGCUUCUGUUAGCCACUGAUSEQ ID GCUUCUGUUAGCCACUGAUUAA NO 396 NO 421 SEQ IDUCAGCUUCUGUUAGCCACUGAUU SEQ ID AGCUUCUGUUAGCCACUGAUUAA NO 397 NO 422SEQ ID UUCAGCUUCUGUUAGCCACUGAUU SEQ ID GCUUCUGUUAGCCACUGAUUAAA NO 398NO 423 SEQ ID UCAGCUUCUGUUAGCCACUGAUUA SEQ ID AGCUUCUGUUAGCCACUGAUUAAANO 399 NO 424 SEQ ID UUCAGCUUCUGUUAGCCACUGAUA SEQ IDGCUUCUGUUAGCCACUGAUUAAA NO 400 NO 425 SEQ ID UCAGCUUCUGUUAGCCACUGAUUAASEQ ID CCAUUUGUAUUUAGCAUGUUCCC NO 401 NO 426 SEQ IDUUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID AGAUACCAUUUGUAUUUAGC NO 402 NO 427SEQ ID UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID GCCAUUUCUCAACAGAUCU NO 403NO 428 SEQ ID UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID GCCAUUUCUCAACAGAUCUGUCANO 404 NO 429 SEQ ID CAGCUUCUGUUAGCCACUG SEQ ID AUUCUCAGGAAUUUGUGUCUUUCNO 405 NO 430 SEQ ID CAGCUUCUGUUAGCCACUGAU SEQ ID UCUCAGGAAUUUGUGUCUUUCNO 406 NO 431 SEQ ID AGCUUCUGUUAGCCACUGAUU SEQ ID GUUCAGCUUCUGUUAGCCNO 407 NO 432 SEQ ID CAGCUUCUGUUAGCCACUGAUU SEQ ID CUGAUUAAAUAUCUUUAUAUCNO 408 NO 433 SEQ ID AGCUUCUGUUAGCCACUGAUUA SEQ ID GCCGCCAUUUCUCAACAGNO 409 NO 434 SEQ ID CAGCUUCUGUUAGCCACUGAUUA SEQ ID GUAUUUAGCAUGUUCCCANO 410 NO 435 SEQ ID AGCUUCUGUUAGCCACUGAUUAA SEQ ID CAGGAAUUUGUGUCUUUCNO 411 NO 436 SEQ ID CAGCUUCUGUUAGCCACUGAUUAA NO 412 DMD Gene Exon 45SEQ ID UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID GUUGCAUUCAAUGUUCUGACAACAG NO 437NO 470 SEQ ID AUUCAAUGUUCUGACAACAGUUUGC SEQ ID UUGCAUUCAAUGUUCUGACAACAGUNO 438 NO 471 SEQ ID CCAGUUGCAUUCAAUGUUCUGACAA SEQ IDUGCAUUCAAUGUUCUGACAACAGUU NO 439 NO 472 SEQ ID CAGUUGCAUUCAAUGUUCUGACSEQ ID GCAUUCAAUGUUCUGACAACAGUUU NO 440 NO 473 SEQ IDAGUUGCAUUCAAUGUUCUGA SEQ ID CAUUCAAUGUUCUGACAACAGUUUG NO 441 NO 474SEQ ID GAUUGCUGAAUUAUUUCUUCC SEQ ID AUUCAAUGUUCUGACAACAGUUUGC NO 442NO 475 SEQ ID GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID UCAAUGUUCUGACAACAGUUUGCCGNO 443 NO 476 SEQ ID AUUGCUGAAUUAUUUCUUCCCCAGU SEQ IDCAAUGUUCUGACAACAGUUUGCCGC NO 444 NO 477 SEQ ID UUGCUGAAUUAUUUCUUCCCCAGUUSEQ ID AAUGUUCUGACAACAGUUUGCCGCU NO 445 NO 478 SEQ IDUGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID AUGUUCUGACAACAGUUUGCCGCUG NO 446 NO 479SEQ ID GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID UGUUCUGACAACAGUUUGCCGCUGC NO 447NO 480 SEQ ID CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID GUUCUGACAACAGUUUGCCGCUGCCNO 448 NO 481 SEQ ID UGAAUUAUUUCUUCCCCAGUUGCAU SEQ IDUUCUGACAACAGUUUGCCGCUGCCC NO 449 NO 482 SEQ ID GAAUUAUUUCUUCCCCAGUUGCAUUSEQ ID UCUGACAACAGUUUGCCGCUGCCCA NO 450 NO 483 SEQ IDAAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID CUGACAACAGUUUGCCGCUGCCCAA N0451 NO 484SEQ ID AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID UGACAACAGUUUGCCGCUGCCCAAU NO 452NO 485 SEQ ID UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID GACAACAGUUUGCCGCUGCCCAAUGNO 453 NO 486 SEQ ID UAUUUCUUCCCCAGUUGCAUUCAAU SEQ IDACAACAGUUUGCCGCUGCCCAAUGC NO 454 NO 487 SEQ ID AUUUCUUCCCCAGUUGCAUUCAAUGSEQ ID CAACAGUUUGCCGCUGCCCAAUGCC NO 455 NO 488 SEQ IDUUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID AACAGUUUGCCGCUGCCCAAUGCCA NO 456 NO 489SEQ ID UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID ACAGUUUGCCGCUGCCCAAUGCCAU NO 457NO 490 SEQ ID UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID CAGUUUGCCGCUGCCCAAUGCCAUCNO 458 NO 491 SEQ ID CUUCCCCAGUUGCAUUCAAUGUUCU SEQ IDAGUUUGCCGCUGCCCAAUGCCAUCC NO 459 NO 492 SEQ ID UUCCCCAGUUGCAUUCAAUGUUCUGSEQ ID GUUUGCCGCUGCCCAAUGCCAUCCU NO 460 NO 493 SEQ IDUCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID UUUGCCGCUGCCCAAUGCCAUCCUG NO 461 NO 494SEQ ID CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID UUGCCGCUGCCCAAUGCCAUCCUGG NO 462NO 495 SEQ ID CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID UGCCGCUGCCCAAUGCCAUCCUGGANO 463 NO 496 SEQ ID CCAGUUGCAUUCAAUGUUCUGACAA SEQ IDGCCGCUGCCCAAUGCCAUCCUGGAG NO 464 NO 497 SEQ ID CAGUUGCAUUCAAUGUUCUGACAACSEQ ID CCGCUGCCCAAUGCCAUCCUGGAGU NO 465 NO 498 SEQ IDAGUUGCAUUCAAUGUUCUGACAACA SEQ ID CGCUGCCCAAUGCCAUCCUGGAGUU NO 466 NO 499SEQ ID UCC UGU AGA AUA CUG GCA UC SEQ ID UGUUUUUGAGGAUUGCUGAA NO 467NO 500 SEQ ID UGCAGACCUCCUGCCACCGCAGAUUCA SEQ ID UGUUCUGACAACAGUUUGCCGCUNO 468 NO 501 GCCCAAUGCCAUCCUGG SEQ ID UUGCAGACCUCCUGCCACCGCAGAUUCNO 469 AGGCUUC DMD Gene Exon 55 SEQ ID CUGUUGCAGUAAUCUAUGAG SEQ IDUGCCAUUGUUUCAUCAGCUCUUU NO 502 NO 505 SEQ ID UGCAGUAAUCUAUGAGUUUC SEQ IDUCCUGUAGGACAUUGGCAGU NO 503 NO 506 SEQ ID GAGUCUUCUAGGAGCCUU SEQ IDCUUGGAGUCUUCUAGGAGCC NO 504 NO 507 DMD Gene Exon 57 SEQ IDUAGGUGCCUGCCGGCUU SEQ ID CUGAACUGCUGGAAAGUCGCC NO 508 NO 510 SEQ IDUUCAGCUGUAGCCACACC SEQ ID CUGGCUUCCAAAUGGGACCUGAA NO 509 NO 511 AAAGAACDMD Gene Exon 59 SEQ ID CAAUUUUUCCCACUCAGUAUU SEQ IDUCCUCAGGAGGCAGCUCUAAAU NO 512 NO 514 SEQ ID UUGAAGUUCCUGGAGUCUU NO 513DMD Gene Exon 62 SEQ ID UGGCUCUCUCCCAGGG SEQ ID GGGCACUUUGUUUGGCG NO 515NO 517 SEQ ID GAGAUGGCUCUCUCCCAGGGACCCUGG NO 516 DMD Gene Exon 63 SEQ IDGGUCCCAGCAAGUUGUUUG SEQ ID GUAGAGCUCUGUCAUUUUGGG NO 518 NO 520 SEQ IDUGGGAUGGUCCCAGCAAGUUGUUUG NO 519 DMD Gene Exon 65 SEQ IDGCUCAAGAGAUCCACUGCAAAAAAC SEQ ID UCUGCAGGAUAUCCAUGGGCUGGUC NO 521 NO 523SEQ ID GCCAUACGUACGUAUCAUAAACAUUC NO 522 DMD Gene Exon 66 SEQ IDGAUCCUCCCUGUUCGUCCCCUAUUAUG NO 524 DMD Gene Exon 69 SEQ IDUGCUUUAGACUCCUGUACCUGAUA NO 525 DMD Gene Exon 75 SEQ IDGGCGGCCUUUGUGUUGAC SEQ ID CCUUUAUGUUCGUGCUGCU NO 526 NO 528 SEQ IDGGACAGGCCUUUAUGUUCGUGCUGC NO 527

The invention claimed is:
 1. An isolated antisense oligonucleotide whosebase sequence consists of 5′-UUCCAACUGGGGACGCCUCUGUUCC-3′ (SEQ ID NO:299), wherein the oligonucleotide comprises a modification.
 2. Theisolated antisense oligonucleotide of claim 1, wherein the modificationcomprises at least one nucleotide analogue, wherein the nucleotideanalogue comprises a modified sugar moiety, a modified backbone, amodified internucleoside linkage, or a modified base, or a combinationthereof.
 3. The isolated antisense oligonucleotide of claim 1, whereinthe modification comprises a modified sugar moiety.
 4. The isolatedantisense oligonucleotide of claim 3, wherein the modified sugar moietyis mono- or di-substituted at the 2′, 3′ and/or 5′ position.
 5. Theisolated antisense oligonucleotide of claim 4, wherein the modifiedsugar moiety comprises a 2′-O-methyl ribose.
 6. The isolated antisenseoligonucleotide of claim 1, wherein the modification comprises amodified backbone.
 7. The isolated antisense oligonucleotide of claim 6,wherein the modified backbone comprises a morpholino backbone, acarbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxidebackbone, a sulfone backbone, a formacetyl backbone, a thioformacetylbackbone, a methyleneformacetyl backbone, a riboacetyl backbone, analkene containing backbone, a sulfamate backbone, a sulfonate backbone,a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazinobackbone or an amide backbone, or a combination thereof.
 8. The isolatedantisense oligonucleotide of claim 7, wherein the modified backbonecomprises a morpholino backbone.
 9. The isolated antisenseoligonucleotide of claim 1, wherein the modification comprises amodified internucleoside linkage.
 10. The isolated antisenseoligonucleotide of claim 9, wherein the modified internucleoside linkagecomprises a phosphorothioate linkage.
 11. The isolated antisenseoligonucleotide of claim 1, wherein the modification comprises amodified base.
 12. The isolated antisense oligonucleotide of claim 1,wherein the oligonucleotide comprises a morpholino ring, aphosphorodiamidate internucleoside linkage, a peptide nucleic acid, alocked nucleic acid (LNA), or a combination thereof.
 13. The isolatedantisense oligonucleotide of claim 1, wherein the oligonucleotidecomprises a 2′-O-methyl phosphorothioate ribose.
 14. The isolatedantisense oligonucleotide of claim 1, wherein the oligonucleotidecomprises a phosphorodiamidate morpholino oligomer (PMO).